RESEARCH ARTICLE Evaluation of Susceptibility and Malaria Vector Potential of annularis s.l. and Anopheles vagus in Assam, India

Sunil Dhiman*, Kavita Yadav, Bipul Rabha, Diganta Goswami, S. Hazarika, Varun Tyagi

Division of Medical Entomology, Defence Research Laboratory, Tezpur, Assam, 784001, India

* [email protected]

Abstract

During the recent past, development of DDT resistance and reduction to pyrethroid suscep- tibility among the malaria vectors has posed a serious challenge in many Southeast Asian countries including India. Current study presents the susceptibility and knock- down data of field collected Anopheles annularis sensu lato and An. vagus spe- cies from endemic areas of Assam in northeast India. Anopheles annularis s.l. and An.

OPEN ACCESS vagus adult females were collected from four randomly selected sentinel sites in Orang pri- mary health centre (OPHC) and Balipara primary health centre (BPHC) areas, and used for Citation: Dhiman S, Yadav K, Rabha B, Goswami D, Hazarika S, Tyagi V (2016) Evaluation of Insecticides testing susceptibility to DDT, malathion, deltamethrin and lambda-cyhalothrin. After insecti- Susceptibility and Malaria Vector Potential of cide susceptibility tests, mosquitoes were subjected to VectorTest™ assay kits to detect Anopheles annularis s.l. and Anopheles vagus in the presence of malaria sporozoite in the mosquitoes. An. annularis s.l. was completely sus- Assam, India. PLoS ONE 11(3): e0151786. ceptible to deltamethrin, lambda-cyhalothrin and malathion in both the study areas. An. doi:10.1371/journal.pone.0151786 vagus was highly susceptible to deltamethrin in both the areas, but exhibited reduced sus- Editor: Richard Paul, Institut Pasteur, FRANCE ceptibility to lambda-cyhalothrin in BPHC. Both the species were resistant to DDT and Received: January 6, 2016 showed very high KDT50 and KDT99 values for DDT. Probit model used to calculate the

Accepted: March 3, 2016 KDT50 and KDT99 values did not display normal distribution of percent knock-down with < Published: March 24, 2016 time for malathion in both the mosquito species in OPHC (p 0.05) and An. vagus in BPHC (χ2 = 25.3; p = 0.0), and also for deltamethrin to An. vagus in BPHC area (χ2 = 15.4; p = Copyright: © 2016 Dhiman et al. This is an open access article distributed under the terms of the 0.004). Minimum infection rate (MIR) of Plasmodium sporozoite for An. vagus was 0.56 in Creative Commons Attribution License, which permits OPHC and 0.13 in BPHC, while for An. annularis MIR was found to be 0.22 in OPHC. Resis- unrestricted use, distribution, and reproduction in any tance management strategies should be identified to delay the expansion of resistance. medium, provided the original author and source are Testing of field caught Anopheles vectors from different endemic areas for the presence of credited. malaria sporozoite may be useful to ensure their role in malaria transmission. Data Availability Statement: All relevant data are available in the paper and its Supporting Information files.

Funding: The authors received no specific funding for this work.

Competing Interests: The authors have declared that no competing interests exist.

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 1/13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

Introduction In the recent years, scaling-up of long-lasting insecticidal nets (LLINs) and to some extent indoor residual spraying (IRS) using insecticides has been a pivotal element in mosquito con- trol strategies. However, rapid emergence and geographical spread of insecticide resistance among malaria vectors has threatens the intervention programmes in many endemic Afro- Asian countries. Only four insecticide classes, which share two modes of action have been approved by the World Health Organization (WHO) for use in mosquito control programmes [1]. Since there are limited number of insecticide groups available, the options to switch over to comparatively more effective insecticide in control operations are restricted. Considering the importance of insecticides in malaria control, regular monitoring of insecticides susceptibility among Anopheles vectors is essential, primarily in the regions where malaria in endemic and subsistently remains a burden to the ethnic communities [2–4]. In India, synthetic pyrethroids have been widely used in LLINs, while DDT is used for IRS in many malaria endemic regions including northeastern states of India. However, few recent studies have indicated considerable level of resistance among some well established malaria vectors against synthetic pyrethroids and DDT in different states of India [5–8]. The northeast region of India is geographically isolated and shares international frontiers on three sides with malaria affected countries like Bangladesh, Bhutan and Myanmar. In this region, although Anopheles minimus and An. dirus are considered as major malaria vectors [5, 9, 10], but recently the abundance of these two mosquito species has decreased [11], while An. annularis and An. vagus became increasingly important due to their high density during the peak malaria season and possible role in malaria transmission. Although both these species are primarily zoophilic, exophilic and exophagic, but also found feeding on human blood and maintaining malaria transmission in the plain areas of northeast India and adjoining Bangladesh [6, 9, 12, 13]. Anopheles annularis Van der Wulp, 1884 is widespread in many Asian countries and recently emerged as an important malaria vector in India and neighbouring countries [6, 10, 14, 15]. An. vagus Doenitz, 1902 is extensively recorded in malaria endemic areas of Indian sub-continent and plays important role in malaria transmission in Bangladesh, Laos and Cam- bodia [12, 13, 16, 17]. Previous studies have shown resistance to DDT and reduced susceptibil- ity to deltamethrin in An. annularis [6], but none of the study has recorded the insecticide resistance status of An. vagus in northeast India. Although An. vagus is a well known malaria vector in many countries now, but in India, no study has demonstrated its potential role in malaria transmission. The objective of the present study was to investigate the insecticide susceptibility of An. annularis s.l. and An. vagus in malaria endemic Udalguri district and Sonitpur district of Assam in northeast India. Since organochloride (OC), synthetic pyrethroids (SP) and organo- phosphate (OP) insecticides are used in malaria control intervention in the region, we have used DDT, deltamethrin, lambda-cyhalothrin and malathion as test insecticides in this study. Furthermore, VectorTest™ malaria sporozoite antigen panel assay has been used for the detec- tion of Plasmodium circumsporozoite antigens in both the mosquitoes species.

Materials and Methods Study area Current study was conducted during March 2013 to August 2013 (pre-monsoon and monsoon season) in malaria endemic Udalguri and Sonitpur districts of Assam state of northeast India. In Udalguri district, four sentinel sites each were randomly selected in Orang primary health

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 2/13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

centre (OPHC) (26° 33’–26° 56’ N to 92° 07’–92° 22’ E), while in Sonitpur district, same num- ber of sentinel sites were chosen in Balipara primary health centre area (BPHC) (26° 41’–27° 02’ N to 92° 38’–92° 59’ E) (Fig 1). The study districts are dominated by socio-economically backward ethnic tribes engaged mainly in tea based agriculture [4]. The climate is humid with an average annual rainfall of about 2,000 mm and temperature varying between 13.5°C to 35.0°C. Study area has many small rivers, duck rearing ponds, spread of tea gardens, vast paddy fields and forests, which provide sufficient breeding habitat for mosquitoes. Both the primary health centres report high incidence of malaria annually [4,18–20]. During the study year OPHC area reported malaria parasite slide positivity rate (SPR) of 2.40 and annual para- sitic index (API) of 3.76, whereas BPHC area reported SPR and API of 2.12 and 0.24 respec- tively. Insecticides use has been intensive with several rounds of spray per growing season due to severe damage of tea and rice by pests. Synthetic pyrethroids are most commonly used in agriculture, whereas the use of organophosphate and carbamate based insecticides is comparatively less common. No specific permissions were required for conducting this activity in both the study areas. We have made the collection of mosquitoes only and none of the study in this research involved the collection and use of rare/endangered/protected species.

Mosquito collection, identification and resistance bioassay Adult indoor resting mosquitoes were collected from the human houses during 0500–600 hours using suction tube and torch light. Mosquitoes were identified morphologically using standard keys used for the identification of medically important Anopheles mosquitoes. The study areas were subjected to a round of indoor residual spray of DDT in February 2013. Healthy and unfed adult females of An. annularis s.l. and An. vagus were exposed to World Health Organisation (WHO) insecticide pre-impregnated papers of DDT (4%), deltamethrin (0.05%), lambda-cyhalothrin (0.05%) and malathion (5%) obtained from University Sains Malaysia, Malaysia in WHO insecticide susceptibility evaluation test kits [21,22]. The control tests were performed using pre-impregnated paper with silicone oil (deltamethrin and lambda- cyhalothrin control), risella oil (DDT control) and olive oil (malathion control) along with each set of insecticide bioassay. Each time 10–15 mosquitoes were used in the test for 1 hour and cumulative knock-down was recorded after an interval of 10 minutes [6]. The mosquitoes were then transferred into the holding tube and fed on 5% sucrose solution. Mortality was recorded after a 24 hour holding period and the resistance status was defined according to WHO guidelines, which state that 98–100% mortality indicates susceptibility, 90–98% indi- cates the possibility of resistance that needs to be confirmed and <90% indicates resistance [23]. After the completion of each test, mosquitoes were re-identified to avoid any error and stored in labeled eppendorf tubes for malaria sporozoite antigen detection assay using Vec- torTest™ assay kit according to the standard manufacturer’s instruction.

Malaria sporozoite antigen assay The adult female An. annularis s.l. and An. vagus were subjected to VectorTest™ assay kits (Vector Test System Inc., CA) to detect the presence of malaria sporozoite in the mosquitoes. For each test a pool of 20–25 mosquitoes of a species was put into the grinding tube provided with the assay kit and homogenised in grinding solution using plastic pestle. The tests were performed according to the standard manufacturer’s instruction provided with the assay kit. The VectorTest™ malaria sporozoite antigen assay is a highly specific and rapid immunochro- matographic test for qualitative determination of circumsporozoite antigens of P. falciparum, P. vivax 210 and P. vivax 247 malaria parasite species in infected mosquitoes [24,25].

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 3/13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

Fig 1. Study area map. Map showing Balipara and Orang primacy health centre (BPHC and OPHC) where mosquito collections were performed for WHO insecticide resistance test. doi:10.1371/journal.pone.0151786.g001 Data analysis The mortality obtained in the mosquito species was corrected using Schneider-Orelli's formula

[26]. Knock-down time (KDT50 and KDT99) along with slope and 95% confidence interval (CI) were determined using Ldp Line computer programme. Chi-square (χ2) test was used to analyse the fitment of probit, while liner regression was used to evaluate if data deviate from linearity.

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 4/13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

Results Insecticide resistance bioassay A total of 1,566 An. annularis s.l. and 1,998 An. vagus mosquitoes were tested in the present study to determine the susceptibility against insecticides (Tables 1 and 2). As per WHO guide- lines [23], the mortality ranging between 98–100% indicates susceptibility, 90–97% as tolerant for which further investigation is needed, and <90% is considered resistant where pre-emptive action is required to manage the resistance against insecticides used for malaria vector control. Based on these recommendations, An. annularis s.l. was completely susceptible to deltame- thrin, lambda-cyhalothrin and malathion in both the study PHCs as the corrected mortality observed was 100% (95% CI- 97.5–100.0), 99.3% (95% CI- 96.3–99.9) and 98.7% (95% CI- 95.3–99.6) respectively in OPHC (Table 1), while 98.1% (95% CI- 95.8–99.2), 98.6% (95% CI- 94.9–99.6) and 98.9% (95% CI- 96.1–99.7) respectively for the three insecticides in BPHC area (Table 2). An. annularis s.l. from both the study areas showed complete resistance to DDT and the corrected mortality recorded was below 75.2% (95% CI- 68.2–82.1). An. vagus mosquitoes were highly susceptible to deltamethrin but exhibited considerably reduced susceptibility to lambda-cyhalothrin in both the areas. The mortality of An. vagus to DDT was recorded below 83.3% (95% CI- 95.3–99.6) in the present study which indicated a high level of resistance to DDT. Against malathion, An. vagus mosquitoes were susceptible in OPHC (corrected mortal- ity- 99.3%; 95% CI- 96.3–99.9), while suspected to be resistant in BPHC as the corrected mor- tality was found to be 97.6% (95% CI- 95.4–98.8) (Tables 1 and 2).

Knock-down effect The knock-down effect of four insecticides determined against An. vagus and An. annularis s.l. in OPHC and BPHC over an exposure time period of one hour has been shown in Tables 1 and 2, whereas the percent knock-down achieved in both the locations has been depicted in Fig 2

(S1 Table) and Fig 3 (S2 Table) respectively. In An. annularis s.l., the KDT50 ranged from 24.7 to 25.3 minutes, while KDT99 ranged from 129.0 to 144.0 minutes during the study. Among all the tested insecticides, least knock-down percent of both the mosquito species (range—35.3– 41.3%) was recorded for DDT, whereas highest knock-down percent ranging from 86.8 to 98.1 was observed for deltamethrin within one hour of exposure time. Both the mosquito species

displayed very high KDT50 and KDT99 values for DDT in both the study locations. The KDT50 values of malathion and lambda-cyhalothrin ranged from 37.7 to 51.2 and 24.5 to 31.8 minutes

respectively. Presently the probit model used to calculate the KDT50 and KDT99 values dis- played normal distribution of percent knock-down with time for all the insecticides except malathion for both the mosquito species in OPHC (p<0.05) and An. vagus in BPHC (χ2 = 25.3; p = 0.0), and also for deltamethrin exposure to An. vagus in BPHC area (χ2 = 15.4; p = 0.004).

Malaria sporozoite antigen assay In the present study, a total of 59 pools (N = 1,340) of female An. vagus and 39 pools (N = 780) of An. annularis s.l were tested for the presence of Plasmodium sporozoite antigen (S3 Table). For An. vagus, 3 pools were found positive in OPHC, whereas 1 pool was positive in BPHC area. Minimum infection rate (MIR) of Plasmodium sporozoite for An. vagus was found to be 0.56 in OPHC and 0.13 in BPHC. On the other hand only 1 pool was found positive for An. annularis in OPHC with a MIR of 0.22 and pool positive rate of 4.35 (Table 3). All the tested mosquito pools found positive for Plasmodium sporozoites corresponded to malaria parasite .

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 5/13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

Table 1. Toxicity and knock-down time of An. annularis and An. vagus in Orang primary health centre (OPHC) area.

2 Insecticide (N) Mosquito %KD1h KDT50 (95% CI) KDT99 (95% CI) Slope χ (p) r CM24 h (95% species (N) ±SD CI) Deltamethrin An. annularis 90.7 (136) 24.7 (22.9–26.5) 129.0 (108.6–160.4) 3.2±0.2 1.9 (0.7) 1 100 (97.5– (150) 100.0) DDT (150) 41.3 (62) 99.2 (71.7–181.4) 14960.1 (3210.2– 1.1±0.2 1.1 (0.9) 1 75.2 (68.6– 319454.1) 82.1) Malathion (150) 62 (93) 51.2 (43.0–73.3) 384.7 (341.7–1583.1) 2.7±0.2 14.7 0.9 99.3 (96.3– (0.005) 99.9) L-cyhalothrin 90 (135) 24.5 (22.8–26.1) 116.7 (99.6–142.2) 3.4±0.2 1.8 (0.8) 1 98.7 (95.3– (150) 99.6) Deltamethrin An. vagus 96 (144) 22.1 (20.6–23.7) 102.4 (88.4–122.8) 3.5±0.2 8.0 (0.08) 1 98.7 (95.3– (150) 99.6) DDT (150) 35.3 (53) 159.5 (100.2– 33625.9 (5099.1– 1.0±0.2 4.1 (0.3) 0.9 83.3 (76.6– 421.3) 2039497.9) 88.5) Malathion (150) 68 (102) 41.2 (34.9–50.9) 252.5 (203.4–566.9) 2.9±0.2 11.7 (0.02) 1 99.3 (96.3– 99.9) L-cyhalothrin 90 (135) 25.2 (23.6–26.8) 109.1 (94.2–131.0) 3.7±0.2 2.6 (0.6) 1 97.3 (93.3– (150) 98.9)

KDT—knock-down time in minutes; CM—corrected mortality in percent; N- number, CI—confidence interval, SD—standard deviation, r—correlation coefficient

doi:10.1371/journal.pone.0151786.t001

Discussion Presently, WHO insecticide bioassays were performed on An. annularis s.l. and An. vagus mos- quitoes to assess their susceptibility to DDT, deltamethrin, malathion and lambda-cyhalothrin in two endemic districts of Assam in northeast India. Different level of susceptibility to the tested insecticides has been observed in the study. WHO recommends the use of 2–3 days old female mosquitoes for insecticide bioassay, however currently field collected mosquitoes repre- senting natural age-structured populations were tested to determine the resistance status. Hence there was a mix of mosquitoes of different age, which probably produced higher mortal- ity than expected by using young mosquitoes. Previous studies have reported that as compared to the young mosquitoes, the level of detoxifying enzymes, namely GST and monooxygenase often decreases with age, leading to an increase in the insecticide susceptibility level [17, 27, 28].

Table 2. Toxicity and knock-down time of An. annularis and An. vagus in Balipara primary health centre (BPHC) area.

2 Insecticide (N) Mosquito species %KD1h (N) KDT50 (95% CI) KDT99 (95% CI) Slope±SD χ (p) r CM24 h (95% CI) Deltamethrin (272) An. annularis 86.8 (236) 25.3 (23.9–26.7) 144.0 (124.8–170.9) 3.1±0.2 4.0 (0.9) 1 98.1 (95.8–99.2) DDT (373) 37.3 (139) 91.4 (79.8–109.3) 1357.3 (841.5–2592.5) 2.0±0.2 3.1 (0.5) 1 65.1 (61.3–70.8) Malathion (139) 79.9 (111) 37.7 (35.4–40.3) 170.8 (139.6–222.6) 3.5±0.3 6.6 (0.2) 1 98.6 (94.9–99.6) L-cyhalothrin (182) 80.8 (147) 31.8 (29.6–34.1) 231.3 (182.8–313.1) 2.7±0.2 4.7 (0.3) 1 98.9 (96.1–99.7) Deltamethrin (424) An. vagus 98.1 (416) 20.5 (18.1–22.7) 85.8 (76.4–105.9) 3.7±0.1 15.4 (0.004) 1 99.1 (97.6–99.6) DDT (326) 36.5 (119) 105.6 (87.6–136.8) 2831.6 (1444.8–7437.6) 1.6±0.1 3.6 (0.5) 1 70.0 (65.1–74.9) Malathion (335) 90.7 (304) 38.6 (34.8–42.5) 91.9 (86.1–115.6) 6.2±0.3 25.3 (0.0) 1 97.6 (95.4–98.8) L-cyhalothrin (313) 83.1 (260) 31.1 (29.8–32.5) 147.2 (129.7–171.1) 3.4±0.2 0.3 (1.0) 1 88.6 (84.9–91.9)

KDT—knock-down time in minutes; CM—corrected mortality in percent; N- number, CI—confidence interval, SD—standard deviation, r—correlation coefficient

doi:10.1371/journal.pone.0151786.t002

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 6/13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

Fig 2. Knock-down rate for different insecticides during 1 hour of exposure in Orang primary health centre (OPHC) area. An. vagus (A), An. annularis (B). DM- deltamethrin, MA- malathion, LC- lambda- cyhalothrin. doi:10.1371/journal.pone.0151786.g002

The results demonstrated that both the mosquito species displayed a high level of biological resistance to DDT as the corrected mortality ranged from 83.3% (95% CI = 76.6–88.5) to as low as 65.1% (95% CI = 61.3–70.8) during the study. Although DDT is extensively used in pub- lic health programmes, but resistance to DDT is widespread among many efficient mosquito – – vectors in different parts of India [6 8, 29 31]. The KDT50 and KDT99 values were also found to be very high and never recorded below 91.4 minutes (95% CI = 79.8–109.3), suggesting that the tested mosquitoes were not much knock-down sensitive to DDT. However, relatively low value of KDT has been recorded in the regions where mosquitoes are susceptible to DDT, whereas high KDT values have been recorded from the regions which reported high level of DDT resistance [6, 29, 32]. DDT is most accepted insecticide in India and its use for many decades now has resulted in high selection pressure and widespread of insecticide resistance. Although kdr mutations have been reported to confer resistance to DDT, but a recent study has again raised this concern by suggesting that about 30% of phenotypically resistant mosqui- toes did not present kdr mutations [33]. There was complete susceptibility to deltamethrin as the corrected mortalities recorded were above 98.1% for both the mosquito species in both the study areas. However a reduced sensitivity was observed for lambda-cyhalothrin in An. vagus as the corrected mortality – – observed ranged from 88.6 (95% CI = 84.9 91.9) to 97.3% (95% CI = 93.3 98.9). The KDT50 and KDT99 values for deltamethrin and lambda-cyhalothrin were comparable for both the

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 7/13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

Fig 3. Knock-down rate for different insecticides during 1 hour of exposure in Balipara primary health centre (BPHC) area. An. vagus (A), An. annularis (B). DM- deltamethrin, MA- malathion, LC- lambda- cyhalothrin. doi:10.1371/journal.pone.0151786.g003

species, but interestingly these values for lambda-cyhalothrin in An. vagus were found to be 1.5

(KDT50) and 1.7 (KDT99) fold high than deltamethrin in BPHC area. Synthetic pyrethroids including deltamethrin and lambda-cyhalothrin are widely used in various public health programmes to control mosquitoes in many countries. However, in the recent years efficacy of these insecticides against potential malaria vectors has been found to reduced in endemic areas [2, 3, 6, 17, 34]. The present study area has vast paddy fields and large scale vegetable cultivation throughout the year, and the pyrethroids are widely used in the control of agricultural pests. Furthermore, the pyrethroid based long lasting insecticidal nets

Table 3. Plasmodium sporozoite detection using VectorTest™ panel assay.

Species Location Pool (n) N Positive MIR PPR An. vagus Orang 27 (20) 540 3 0.56 11.11 Balipara 32 (25) 800 1 0.13 3.13 An. annularis Orang 23 (20) 460 1 0.22 4.35 Balipara 16 (20) 320 0 0.00 0.00 Total 98 2120 5 0.24 5.10

N—total number tested; n—number in each pool; MIR—minimum infection rate; PPR—pool positive rate doi:10.1371/journal.pone.0151786.t003

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 8/13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

(LLINs) have been considered as the cornerstone of malaria control programmes and distrib- uted free of cost by the government agencies in the recent years. All these activities have increased the selection in malaria vectors to this class of insecticides. A significant level of resis- tance to pyrethroid was found associated with the agriculture intensity in Africa, indicating that resistance level increases with the increase in agriculture spread [2, 35]. Presently, the

tested mosquito were knock-down sensitive to pyrethroid as the KDT50 an KDT99 values were considerably lower but found to be higher than achieved for An. annularis previously [6]. In a study conducted in Mekong region, An. vagus was found to be highly knock-down resistant to deltamethrin and revealed the presence of a L1014S kdr mutation [17]. Present results revealed complete susceptibility to malathion except for An. vagus in BPHC area where the mortality recorded was 97.6% (95% CI = 95.4–98.8) indicating reduced suscep- tibility which warrants regular monitoring. Although malathion is extensively used in the con- trol of vector mosquitoes in different endemic regions of India but no study has clearly indicated the development of resistance to malathion [29]. Malathion is mostly used in fogging to control dengue vectors during the epidemics and not in the control of malaria vectors, there- fore the chances of exposing Anopheles mosquitoes to malathion are limited except some acci- dental exposure. Mutations in the voltage gate sodium channel gene have been shown as important mecha- nism conferring high level of cross resistance to DDT and synthetic pyrethroids. Currently no evidence of cross resistance to DDT and pyrethroids has been observed, however the study has underlined the existence of DDT resistance in malaria vectors and possible decline in sensitiv- ity to the synthetic pyrethroids, but does not suggests the mechanism which could be attributed to the problem of resistance. Studies have very well documented the role of target-site muta- tions in insecticide resistance, however these were not found solely responsible for resistance and some detoxifying genes acting in concert with these mutations in voltage gated sodium channels were reported to confer extreme levels of resistance [28, 35–39]. A recent research demonstrated that glutathione S-transferase gene GSTe2 was the most over-expressed detoxifi- cation gene in DDT and permethrin resistant Anopheles funestus mosquitoes [40], whereas another study [41] claimed that mutation L1014F was more efficient in conferring resistance to DDT as compared to pyrethroids, which might be a reason that the mosquitoes in the present study displayed high level of resistance to DDT. Furthermore, the studies have also suggested that kdr may act with certain unidentified co-factors to create resistance phenotype [42] or the resistance could be a multigenic phenomenon [43], thereby unable to fully explain the resis- tance mechanism. A study conducted in Bihar state of India to assess the utility of DDT based indoor residual spray found that sand were susceptible to deltamethrin but high level of resistance was observed to DDT [44]. In the present study, altogether five pools out of total 98 pools were detected positive using VectorTest™ for the presence of Plasmodium antigen suggesting that both these species play important role in malaria transmission. Four pools of An. vagus were found positive for Plas- modium falciparum revealing that An. vagus might be playing crucial role as malaria vector in the study area. During the past few years An. vagus has emerged as an important malaria vec- tor, reported in large number in India and neighbouring countries [10,12] and incriminated as vector of malaria in India [45] and Bangladesh [12, 13]. An. annularis s.l. although found posi- tive for Plasmodium in only one pool but regarded as important malaria vector in many endemic regions of India [6, 9]. In the present study primary malaria vectors An. minimus and An. dirus did not encounter, however recognised malaria vectors An. culicifacies (N = 68) and An. fluviatilis (N = 14) were recorded in low density and only one pool belonging to An. culici- facies s.l. in BPHC was found positive for Plasmodium falciparum sporozoite (MIR = 1.6). A recent investigation conducted in northeast India has revealed that 21.1% of the wild collected

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 9/13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

An. annularis were fed on human blood, while 2.6% were found positive for Plasmodium falcip- arum malaria parasite [6]. VectorTest™ antigen panel assay has been found effective in moni- toring the disease spread by detecting malaria parasite in the wild collected mosquitoes [24, 25]. The used assay is rapid, one step procedure and qualitatively identifies specific peptide epi- topes of circumsporozoites of the types of Plasmodium sporozoites. The present results confirm the resistance to DDT and reduced susceptibility of pyrethroid insecticides which could gradually increase and spread into the other areas where complete susceptibility is reported at present. Mosquito control research and comprehensive vector tool development requires thorough analysis of such results and their consequences in a large area. Current study was carried out in two high malaria reporting and logistically accessible areas, however such studies using other malaria vectors should also be conducted in far flung and inaccessible areas which experience considerable toll of malaria related mortality and morbid- ity annually. Large number of mosquito specimen corresponding to well known and all possi- ble malaria vector species are needed to be tested from different areas to get a clear insight about the role of each vector in malaria transmission in northeast India.

Conclusion For the first time field collected An. vagus and An. annularis s.l. mosquitoes using such a large sample size were evaluated against different insecticides in northeast India and found completely resistant to DDT, while completely sensitive to deltamethrin. Lambda-cyhalothrin susceptibility was reduced in An. vagus. Further investigations are recommended to under- stand the mechanism underlying the phenotypical resistance to DDT and declining susceptibil- ity to synthetic pyrethroid in order to guide judicious selection of suitable insecticides for vector control interventions. Resistance management strategies should be identified and con- sidered to delay the expansion of insecticide resistance. Present results strongly advocate that both An. annularis and An. vagus may be playing more important role in malaria transmission than thought previously. Testing of Plasmodium parasite presence in the field caught potential Anopheles vectors prevailing in high density from different areas, in addition to the well estab- lished vectors, could be useful to highlight the role of these little known and practically ignored vectors in malaria transmission.

Supporting Information S1 Table. knock-down of An. annularis and An. vagus in OPHC. (XLS) S2 Table. Knock-down of An. annularis and An. vagus mosquitoes in BPHC. (XLS) S3 Table. Data of Vectortest malaria sporozoite panel assay. (XLS)

Acknowledgments Authors are thankful to Dr Vijay Veer, Ex-Director, Defence Research Laboratory, Tezpur for providing necessary help and technical advice during the entire study. We are grateful to the villagers of the study areas for allowing collection of mosquitoes in the houses during early morning hours. The help and support rendered by the Gaonburhas (village headman) of the study villages and local health workers is deeply acknowledged.

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 10 / 13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

Author Contributions Conceived and designed the experiments: SD KY DG BR. Performed the experiments: SD KY DG BR SH VT. Analyzed the data: SD KY. Contributed reagents/materials/analysis tools: SD SH. Wrote the paper: SD KY SH VT. Involved in the revision of manuscript during the entire review process: KY SD VT DG BR.

References 1. Constant VA, Koudou BG, Jones CM, Weetman D, Ranson H. Multiple-insecticide resistance in Anopheles gambiae mosquitoes, Southern Cote d’Ivoire. Emerg Infec Dis. 2012; 18(9):1508–1511. 2. Agossa FR, Gnanguenon V, Anagonou R, Azondekon R, Aizoun N, Sovi A, et al. Impact of insecticide resistance on the effectiveness of pyrethroid-based malaria vectors control tools in Benin: Decreased toxicity and repellent effect. PLoS One. 2015; 10(12):e0145207. doi: 10.1371/journal.pone.0145207 PMID: 26674643 3. Ndiath MO, Mazenot C, Sokhna C, Trape JF. How the malaria vector Anopheles gambiae adapts to the use of insecticide-treated nets by African populations. PLoS One. 2014; 9(6):e97700. doi: 10.1371/ journal.pone.0097700 PMID: 24892677 4. Yadav K, Dhiman S, Rabha B, Saikia PK, Veer V. Socio-economic determinants for malaria transmis- sion risk in an endemic primary health centre in Assam, India. Infect Dis Pov. 2014; 3:19. 5. Dhiman S, Gopalakrishnan R, Goswami D, Baruah I, Singh L. Malaria epidemiology along Indo-Bangla- desh border in Tripura state, India. South East Asian J Trop Med Pub Hlth. 2010; 41(6):1279–1289 6. Dhiman S, Rabha B, Goswami B, Das NG, Baruah I, Bhola RK, et al. Insecticide resistance and human blood meal preference of Anopheles annularis in Assam Meghalaya border, Northeast India. J Vector Borne Dis. 2014; 51:133–136. 7. Mishra AK, Chand SK, Barik TK, Dua VK, Raghavendra K. Insecticide resistance status in Anopheles culicifacies in Madhya Pradesh, central India. J Vector Borne Dis. 2012; 49:39–41. PMID: 22585243 8. Shetty V, Sanil D, Shetty NJ. Insecticide susceptibility status in three medically important species of mosquitoes, Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus, from Bruhat Bengaluru Mahanagara Palike, Karnataka, India. Pest Manag Sci. 2013; 69(2):257–267. doi: 10.1002/ps.3383 PMID: 22926921 9. Dhiman S, Bhola RK, Goswami D, Rabha B, Kumar D, Baruah I, et al. Polymerase chain reaction detection of human host preference and Plasmodium parasite infections in field collected potential malaria vectors. Pathog Glob Health. 2012; 10(3):177–180. 10. Dev V, Sharma VP. The dominant mosquito vectors of human malaria in India. In: Sylvie Manguin, edi- tor. Anopheles mosquitoes: New insights into malaria vectors. Rijeka, Croatia: In Tech. 2013; 239– 271. 11. Yadav K, Dhiman S, Rabha B, Goswami D, Saikia PK, Veer V. Disappearance of An. minimus and An. dirus from certain malaria endemic areas of Assam, India. J -Borne Dis. 2016; Article in press. Available at http://jad.tums.ac.ir/index.php/jad/article/view/391. 12. Alam MS, Khan MGM, Chaudhury N, Deloer S, Nazib F, Bangali AM, et al. Prevalence of anopheline species and their Plasmodium infection status in epidemic-prone border areas of Bangladesh. Malar J. 2010; 9:15. doi: 10.1186/1475-2875-9-15 PMID: 20074326 13. Bashar K, Tuno K. Seasonal abundance of Anopheles mosquitoes and their association with meteoro- logical factors and malaria incidence in Bangladesh. Parasit Vectors. 2014; 7:442. doi: 10.1186/1756- 3305-7-442 PMID: 25233890 14. Singh RK, Haq S, Kumar G, Dhiman RC. Bionomics and vectorial capacity of Anopheles annularis with special reference to India: a review. J Commun Dis. 2013; 45(1–2):1–16. PMID: 25141549 15. WHO. Anopheline species complexes in South and South-east Asia. World Health Organization, SEARO Tec Pub. 2007; 57:17–19. 16. Rueda LM, Pecor JE, Harrison BA. Updated distribution records for Anopheles vagus (Diptera: Culici- dae) in the Republic of Philippines, and considerations regarding its secondary vector roles in South- east Asia. Trop Biomed. 2011; 28(1):181–187. PMID: 21602785 17. Verhaeghen K, Van Bortel W, Trung H, Sochantha T, Keokenchanh K, Coosemans M. Knockdown resistance in Anopheles vagus, An. sinensis, An. paraliae and An. peditaeniatus populations of the Mekong region. Parasit Vectors. 2010; 3:59. doi: 10.1186/1756-3305-3-59 PMID: 20646327 18. Nath MJ, Bora AK, Yadav K, Talukdar PK, Dhiman S, Baruah I, et al. Prioritizing areas for malaria con- trol using geographical information system in an endemic district of Assam, India. Public Health. 2013; 127:572–580. PMID: 23701814

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 11 / 13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

19. Yadav K, Nath MJ, Talukdar PK, Saikia PK, Baruah I, Singh L. Malaria risk areas of Udalguri district of Assam, India: a GIS-based study. Int J Geogr Inf Sci. 2012; 26(1):123–131. 20. Kumar D, Dhiman S, Rabha B, Goswami D, Deka M, Singh L, et al. Genetic polymorphism and amino acid sequence variation in Plasmodium falciparum GLURP R2 repeat region in Assam, India, at an interval of five years. Malar J. 2014; 13:450. doi: 10.1186/1475-2875-13-450 PMID: 25416405 21. WHO. Test procedures for insecticide resistance monitoring in malaria vectors, bio-efficacy and persis- tence of insecticide on treated surfaces. WHO/CDS/CPC/MAL/98.12. 1998; WHO press, Geneva, Switzerland. 22. WHO. Guidelines for testing mosquito adulticides for indoor residual spraying and treatment of mos- quito nets. WHO/CDS/NTD/WHOPES/GCDPP/3. 2006; WHO press, Geneva, Switzerland. 23. WHO. Test procedures for insecticide resistance monitoring in malaria vectors mosquitoes. Geneva, Switzerland: World Health Organization, WHO press 2013; 1–39. 24. Ryan JR, Dave K, Emmerich E, Garcia L, Yi L, Coleman RE, et al. Dipsticks for rapid detection of Plas- modium in vectoring Anopheles mosquitoes. Med Vet Entomol. 2001; 15:225–230. PMID: 11434560 25. Sattabongkot J, Kiattibut C, Kumpitak C, Ponlawat A, Ryan JR, Chan AST, et al. Evaluation of the VecTest malaria antigen panel assay for the detection of Plasmodium falciparum and P. vivax circum- sporozoite protein in anopheline mosquitoes in Thailand. J Med Entomol. 2004; 41:209–214. PMID: 15061280 26. Puntener W. Manual for field trials in plant protection. Second edition. Agricultural Division, Ciba- Geigy Limited, 1981. 27. Chouaibou MS, Chabi J, Bingham GV, Knox TB, N’Dri L, Kesse NB, et al. Increase in susceptibility to insecticides with aging of wild Anopheles gambiae mosquitoes from Cote d’Ivoire. BMC Infect Dis. 2012; 12:214. doi: 10.1186/1471-2334-12-214 PMID: 22974492 28. Xu T, Zhong D, Tang L, Chang X, Fu F, Yan G, et al. Anopheles sinensis mosquito insecticide resis- tance: comparison of three mosquito sample collection and preparation methods and mosquito age in resistance measurements. Parasit Vectors. 2014; 7:54. doi: 10.1186/1756-3305-7-54 PMID: 24472598 29. Yadav K, Rabha B, Dhiman S, Veer V. Multi-insecticide susceptibility evaluation of dengue vectors Ste- gomyia albopicta and St. aegypti in Assam, India. Parasit Vectors. 2015; 8:143. doi: 10.1186/s13071- 015-0754-0 PMID: 25886449 30. Tikar SN, Mendki MJ, Sharma AK, Sukumaran D, Veer V, Prakash S, et al. Resistance status of the malaria vector mosquitoes, Anopheles stephensi and Anopheles subpictus towards adulticides and lar- vicides in arid and semi-arid areas of India. J Insect Sci. 2011; 11:85. doi: 10.1673/031.011.8501 PMID: 21870971 31. Raghavendra K, Verma V, Srivastava HC, Gunasekaran K, Sreedari U, Dash AP. Persistence of DDT, malathion & deltamethrin resistance in Anopheles culicifacies after their sequential withdrawal from indoor residual spraying in Surat district of Gujarat India. Indian J Med Res. 2010; 132:260–264. PMID: 20847371 32. Betson M, Jawara M, Awolola TS. Status of insecticide susceptibility in Anopheles gambiae s.l. from malaria surveillance sites in The Gambia. Malar J. 2009; 8:187. doi: 10.1186/1475-2875-8-187 PMID: 19656399 33. Ndiath MO, Sougoufara S, Gaye A, Mazenot C, Faye O, Sokhna C, et al. Resistance to DDT and pyre- throids and increased kdr mutation frequency in An. gambiae after the implementation of permethrin- treated nets in Senegal. PLoS One. 2012; 7(2):e31943. doi: 10.1371/journal.pone.0031943 PMID: 22384107 34. Dhiman S, Veer V. Culminating anti-malaria efforts at long lasting insecticidal net?. J Inf Pub Hlth. 2014; 7:457–464. 35. Nkya TE, Akhouayri I, Poupardin R, Batengana B, Mosha F, Magesa S, et al. Insecticide resistance mechanisms associated with different environments in the malaria vector Anopheles gambiae: a case study in Tanzania. Malar J. 2014; 13:28 doi: 10.1186/1475-2875-13-28 PMID: 24460952 36. Ranson H, N’Guessan R, Lines J, Moiroux N, Nkuni Z, Corbel V. Pyrethroid resistance in African anopheline mosquitoes: what are the implications for malaria control? Trends Parasitol. 2011; 27 (2):91–98. doi: 10.1016/j.pt.2010.08.004 PMID: 20843745 37. Protopopoff N, Matowo J, Malima R, Kavishe R, Kaaya R, Wright A, et al. High level of resistance in the mosquito Anopheles gambiae to pyrethroid insecticides and reduced susceptibility to bendiocarb in north-western Tanzania. Malar J. 2013; 12:149. doi: 10.1186/1475-2875-12-149 PMID: 23638757 38. Mitchell SN, Rigden DJ, Dowd AJ, Lu F, Wilding CS, Weetman D, et al. Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae. PLoS One. 2014; 9 (3):e92662. doi: 10.1371/journal.pone.0092662 PMID: 24675797

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 12 / 13 Insecticide Susceptibility and Vector Potential of Malaria Vectors

39. Singh OP, Dykes CL, Sharma G, Das MK. L1014F-kdr mutation in Indian Anopheles subpictus (Dip- tera: culicidae) arising from two alternative transversions in the voltage-gated sodium channel and a single PIRA-PCR for their detection. J Med Entomol. 2015; 52(1):24–27. doi: 10.1093/jme/tju013 PMID: 26336276 40. Riveron JM, Yunta C, Ibrahim SS, Djouaka R, Irving H, Menze BD, et al. A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector. Genome Biol. 2014; 15:R27 doi: 10.1186/gb-2014-15-2-r27 PMID: 24565444 41. Nwane P, Etang J, Chouaibou M, Toto JC, Kerah-Hinzoumbe C, Mimpfoundi R, et al. Trends in DDT and pyrethroid resistance in Anopheles gambiae s.s. populations from urban and agro-industrial set- tings in southern Cameroon. BMC Inf Dis. 2009; 9:163. 42. Bass C, Nikou D, Donnelly MJ, Williamson MS, Ranson H, Ball A, et al. Detection of knockdown resis- tance (kdr) mutations in Anopheles gambiae: a comparison of two new high throughput assays with existing methods. Malaria J. 2007; 6:111. 43. Brooke BD. Kdr: Can a single mutation produce an entire insecticide resistance phenotype? Trans Roy Soc Trop Med Hyg. 2008; 102:524–525. doi: 10.1016/j.trstmh.2008.01.001 PMID: 18295809 44. Coleman M, Foster GM, Deb R, Singh RP, Ismail HM, Shivam P, et al. DDT-based indoor residual spraying suboptimal for visceral leishmaniasis elimination in India. PNAS. 2015; 112(28):8573–8578. doi: 10.1073/pnas.1507782112 PMID: 26124110 45. Prakash A, Bhattacharyya DR, Mohapatra PK, Mahanta J. Role of the prevalent Anopheles species in the transmission of Plasmodium falciparum and P. vivax in Assam state, north-eastern India. Ann Trop Med Parasitol. 2004; 98(6):559–68. PMID: 15324463

PLOS ONE | DOI:10.1371/journal.pone.0151786 March 24, 2016 13 / 13